J. Cell Sci. 19, 157-167 (i975) 157 Printed in Great Britain

LOCALIZATION OF IN COTYLEDONS

A. E. CLARKE AND R. B. KNOX School of Botany, University of Melbourne AND M. A. JERMYN CSIRO Division of Chemistry, Parkville 3052 Victoria, Australia

SUMMARY High-resolution techniques for the localization of lectins are described. (Con A) and phytohaemagglutinin (PHA) are localized using a fluorescent method with (FITC)-labelled immunoglobulins which bind to the lectins in sections of jack and red kidney cotyledons. Specificity is defined by the use of specific sugar inhibitors. Both Con A and PHA are found in cytoplasmic sites. Lectins with /?-glycoside specificity are detected with red-coloured artificial carbohydrate antigens. The /?-galactosyl and /9-glucosyl antigens bind specifically to clustera of spherical bodies in the intercellular spaces, to cell wall sites, and to the periphery of the cytoplasm associated with the cell membrane.

INTRODUCTION Plant lectins are soluble or which can be extracted from plants and which interact in a variety of ways with mammalian cells by virtue of their property of specific binding to carbohydrates. The configuration of the carbohydrate moiety required for binding to a has been well defined for a number of lectins (see Lis & Sharon, 1973); and most lectins have multiple carbohydrate-binding sites. A consequence of these properties is that lectins may precipitate glycoproteins and polysaccharides containing accessible sugar sequences which fulfil their specificity requirements. Also, lectins may interact with the carbohydrates of mammalian cell plasma membranes, and with isolated plant protoplasts (Glimelius, Wallin & Eriksson, 1974); this may result in agglutination of the cells, or in certain cases, to changes in the growth pattern of the cells. All these effects can be inhibited by the simple sugars for which the particular lectin is specific. The best known lectin, Concanavalin A (Con A), comprises up to 3 % of the total protein of the jack bean (Canavalia ensiformis): its sugar-binding requirements are known in detail, thus it will bind a-D-mannopyranose, a-D-glucopyranose and steri- cally related structures (So & Goldstein, 1967). The lectin from red kidney bean is known as phytohaemagglutinin (PHA) because of its very potent agglutinating effects on red blood cells. It is also a powerful and both these effects can be inhibited by high concentrations of iV-acetyl-D-galactosamine. Recently, Jermyn (1974) and Jermyn & Yeow (1975) have described a further class of lectins of widespread occurrence in plants, the 'all-/?' lectins. These substances are 158 A. E. Clarke, R. B. Knox and M. A. Jermyn glycoproteins which interact with the /?-glycosyl determinants of artificial carbo- hydrate antigens (Yariv, Rapport & Graf, 1962). These antigens are prepared by coupling diazotized 4-amino phenyl glycosides with phloroglucinol. The resulting products are highly aggregated and provide a red-coloured, high mol. wt. molecule with multiple carbohydrate-determinant groups of a clearly defined configuration. Despite the prominence of plant lectins such as Con A in contemporary cell biology, little is known of the role of these lectins in plants. A number of suggestions have been put forward, and these have been summarized by Lis & Sharon (1973). Lectins may act as plant antibodies produced in response to soil bacteria, or as agents of plant protection from microbial attack through the inhibition of fungal cell-wall-hydrolysing enzymes. Alternatively, they may be involved in sugar transport and storage, or even in the binding of enzymes into batteries of multienzyme systems. The may act as controlling agents of cell differentiation and division, particularly at germination. In approaching this problem, we have suggested that certain lectins may well be involved in the determination of self-recognition between plant cells (Knox, 1975). In order to fulfil such a role, lectins would presumably have to be located at the interface between cells. We have accordingly developed high-resolution methods to locate lectins in plant cells precisely; this may provide an insight into their possible functions.

MATERIALS AND METHODS Source of seeds Jack , Canavalia ensiformis, were obtained from the Genetic Resources section of CSIRO Division of Plant Industry (CPI No. 59505). Red kidney beans, (CPI No. W245), were obtained from Dr R. Biggs, CSIRO Division of Plant Industry; pigeon pea, Cajanus cajan, from Watters & Sons, Melbourne, and lima beans, Phaseolus lunatus cv. Early Thorogreen from CSIRO Division of Plant Industry (CPI No. N934). Seeds were soaked between 2 layers of moist cotton wool for 24-48 h, at room temperature, before sectioning.

Reagents Methyl a-D-glucopyranoside, a-D-galactose, and iV-acetyl-a-D-galactosamine were from Calbiochem, San Diego, Calif., U.S.A. Salicin was from Sigma Chemical Co., St Louis, Mo., U.S.A. Dimethyl sulphoxide was from Unilab, Sydney, Australia. The fluorescein iso- thiocyanate (FITC)-labelled globulin preparation used was from Behringwerke, Marburg-Lahn, Germany (goat anti-rabbit IgG, OTKF 04/05).

Localization of Con A and PHA Both these lectins will precipitate serum glycoproteins in Ouchterlony tests. This has pre- vented us from using high-titre antiserum raised in rabbits against the lectins for immuno- fluorescence localization, since the binding of normal serum components to the lectins obscures any specific antibody binding. However, we have developed a fluorescence technique for the localization of these lectins which relies on binding of the lectins to serum glycoproteins and inhibition of this binding by simple sugars to define staining specificity. We have used a FITC- labelled globulin preparation which was shown to interact with both Con A and PHA in Ouchterlony gel diffusion tests. These interactions were abolished by the presence of methyl a-D-glucoside and JV-acetyl-D-galactosamine respectively, the sugars which fulfil the Lectin localization in cotyledons 159 lectin-binding requirements, but were not affected by the presence of the sugars galactose or a-methyl glucose respectively, which do not fulfil the lectin-binding requirements. Freehand razorblade sections of jack or red kidney bean were placed in the FITC-labelled globulin preparation diluted 1:2 or 1:10 with phosphate-buffered saline (0-15 M NaCl, 005 M phosphate buffer pH 72), for 20 min at room temperature, rinsed in saline for 10 min, and mounted in saline for fluorescence microscopy. As controls of specificity, sections were pre- treated with specific sugars. Jack bean sections were treated for 10 min with 10%, w/v, methyl a-D-glucoside or galactose, red kidney bean sections with 1 %, w/v, A^-acetyl galactos- amine or 10 %, w/v, glucose. Other sections were pretreated in saline in which the sugars were prepared. Sections were then exposed to the labelled IgG containing the same sugar. Fluorescence-microscope observations were carried out using reflected light in the blue region, with a Carl Zeiss reflected-light incident fluorescent illuminator.

Azo-dye technique for localization of (3-lectins Freehand razorblade sections of the various bean seeds were placed in solutions of artificial carbohydrate antigens, prepared as described by Jermyn & Yeow (1975) either fresh or after fixation for 4 or 12 h in 2-5 % glutaraldehyde in phosphate-buffered saline at 4 °C. The fixed material was extensively washed in saline at 4 CC before staining. For these experiments, the /7-glucosyl, /?-galactosyl, a-galactosyl and a-mannosyl derivatives were used. Stock solutions (10 mg/ml) in dimethyl sulphoxide (DMSO) were prepared and diluted 1:10 with phosphate- buffered saline just before use. The final solution contained antigen (1 mg/ml) in 10 % DMSO. Sections were stained for 30 min at room temperature, washed in saline for 10 min, and viewed by bright-field microscopy. As controls of staining specificity, 2 methods were adopted. First, parallel sections were treated with artificial antigens of a-glycosyl as well as /?-glycosyl specificity. Secondly, the effect of a simple /?-glycoside, salicin, on the reaction was examined. Sections of jack bean were placed in a salicin solution (25 % in saline) for 15 min before reaction with the /9-glucosyl antigen in the presence of salicin (2-5%).

OBSERVATIONS Con A has proved to be cytoplasmic in its cellular localization in 24-h-soaked jack bean cotyledons (Fig. IA). It occurs throughout the cotyledons, including the epi- dermal cells which are frequently heavily stained. In the cotyledon parenchyma, the fluorescence is associated with the protein bodies and also the outer surface of the starch grains (Fig. IB). Occasionally, some staining has been found in the cell walls and intercellular spaces, but usually these sites are negative. Binding occurs to the cell walls at the sites of pit fields on the inner faces of the parenchyma cell walls, but such binding also occurs in the control sections incubated with a-methyl glucose. Pretreatment with saline alone or with galactose had no effect on staining, but it was almost completely abolished by methyl a-D-glucoside (Fig. 1 c). PHA localization in red kidney beans has proved to be similar to Con A, in that specific fluorescence is entirely associated with cytoplasmic sites, confirming the observations of Mialonier et al. (1973) using immunoperoxidase methods. JV-acetyl galactosamine pretreatment abolished staining, while pretreatment with a-methyl glucoside had no effect. 160 A. E. Clarke, R. B. Knox and M. A. Jermyn

Localization of fl-lectins Jackbean. Both y?-galactosyl and /?-glucosyl artificial antigens gave similar results, and intense deposits of orange-red reaction product were obtained in the intercellular spaces of the cotyledon parenchyma and at the periphery of the cytoplasm associated with the cell membrane (Fig. 2). No differences in staining patterns could be detected between the fresh and the glutaraldehyde-fixed sections, after either 4 or 12 h fixation. At higher magnification, the intercellular spaces were seen to be filled with clusters of spherical bodies, varying in size up to a maximum of 2-3 /(tn (Fig. 2c). The reaction was also apparent in the cell walls, especially in the central middle lamella zone. Treatment of the cotyledon sections with 2-5% salicin in 0-15 M NaCl for 15 min prior to staining with the Yariv antigen, abolished all staining at the inter- cellular spaces, but did not inhibit the binding at the cell walls (Fig. 3). In a control experiment, pretreatment of the sections in the saline did not affect staining either at the intercellular spaces or at the cell walls. Red kidney bean. Antigen binding in the intercellular spaces was generally found only in the outer parenchyma. Most binding was associated with the greatly thickened and fluted cell walls of the parenchyma cell (Fig. 7). Binding of antigen to sites associated with the circular pit field on the inner wall face were also present in controls. Lima bean. Some binding of antigen was evident in the intercellular spaces, but the most prominent was associated with the fluted margins of the cell walls adjacent to the cell membrane (Fig. 8). Pigeon pea. Similar patterns were obtained, though the intercellular spaces were smaller and the reaction product less easy to detect (Fig. 9). Reaction product was also prominent, apparently in the inner layer of the parenchyma cell walls adjacent to the cytoplasm.

Controls of staining specificity For each tissue, control sections were prepared using both a-galactosyl and a-mannosyl artificial antigens. The a-galactosyl antigen gave no staining in the cyto- plasm or intercellular spaces (Fig. 4) of any of the beans tested except red kidney bean, which showed faint binding to cytoplasmic sites. This is due to binding to PHA which will precipitate a-galactosyl antigen in gel diffusion tests. This confirms the cytoplasmic localization defined by the fluorescent method. Some binding to the pit fields was observed, especially in jack bean. This is interpreted as non-specific staining, since toluidine blue rapidly stains these sites a red-purple colour, indicative of the presence of phenolic complexes (Feder & O'Brien, 1968). The a-mannosyl antigen bound strongly to the cytoplasmic sites of jack bean sections (Fig. 5), but not detectably to any of the other bean sections. Con A binds preferentially to a-D- , so such a result confirms the localization of Con A obtained with the fluorescent method. No reaction product was associated with the cell walls or inter- cellular spaces, so that results were almost the converse of those with the /?-glycosyl antigens. The patterns obtained in these various tests is characteristic of total proteins within Lectin localization in cotyledons 161 the tissue, as seen by staining patterns following incubation in ponceau 2R (Fig. 6) using the method of Flint & Moss (1970). The cytoplasm is intensely stained, with conspicuous staining of material in the intercellular spaces. Similar fresh sections, exposed to the periodic acid-Schiff method for carbohydrate localization, showed staining in the cell walls and starch grains.

DISCUSSION Cellular sites of Con A and PHA Con A localization in plant cells has not previously been reported. The fluorescent method for Con A and PHA localization provides a very sensitive tool that is inde- pendent of non-specific binding problems. Even with very high-titre antisera, we have found it difficult to interpret normal serum control slides in immunofluorescence procedures because of the high level of binding shown by normal serum to both these lectins. The method described in this paper is likely to have wide applications in locating lectin activity during differentiation, and in tissues other than seeds. Using immunoperoxidase methods, Mialonier et al. (1973) detected PHA in the cytoplasm of Phaseolus cotyledon parenchyma, with a suggestion also of some activity associated with the cell walls. Our experiments confirm this predominantly cytoplasmic localization. The fluorescent method has the additional advantage of greater sensitivity and specificity, since peroxidases occur widely in plants and would make the inter- pretation of controls more difficult. Peroxidase, itself a glycoprotein, binds readily to Con A, and this reaction has been used to locate Con A binding to mammalian cell surfaces using enzyme cytochemical techniques to reveal the peroxidase (Bernhard & Avrameas, 1971). However, both in the thick sections used in this study, and on cell surfaces, reflected-light fluorescence microscopy has advantages, since the exciting light does not pass through the tissue. The fluorescence procedure is applicable to a wide range of plant tissues, where the specific fluorescence is clearly differentiated from background autofluorescence. Both Con A and PHA appear, on the present evidence and that of Mialonier et al. (1973), to be located at cytoplasmic sites. This presumably means that their functions are likely to involve cytoplasmic components. It is perhaps significant that Con A is present at greatest activity in developing jack bean cotyledons at the peak of synthesis of storage protein, possibly suggesting some role in the laying down of storage materials. However, it is also present and active in germinating seeds, so a role in the utilization of storage products is also possible.

Extracellular location of fi-lectins A novel feature of this work has been the discovery that lectins with /?-glycoside specificity are located in the intercellular spaces, cell walls, and peripheral cytoplasm of cotyledon parenchyma cells. It is highly unlikely that this localization has been influenced by diffusion artifacts, particularly since toluidine-blue staining has shown that phenolic materials are absent from these particular sites. The possibility that the observed extracellular location of the /Mectin is due to non-specific binding or pre- cipitation of lectin which might have been solubilized in the staining solution is II CEL 19 162 A. E. Clarke, R. B. Knox and M. A. Jermyn excluded, since fixation under conditions which would immobilize the lectin does not alter the staining pattern. Opik (1966) investigated the fine structure of germinating Phaseolus cotyledons, and observed electron-lucent vesicles frequently filling the intercellular spaces. The sizes of these vesicles correspond well to the clusters of bodies staining for /Mectin activity in jack bean and red kidney bean. The nature of these spherical structures needs to be fully investigated cytochemically during seed development, and the jack bean would be ideal material. A question of particular interest is whether these extracellular materials originate from the cells surrounding the intercellular channels, or whether they may be products of special secretory cells located elsewhere in the cotyledon. This is of some importance, since Yomo & Taylor (1973) have shown that proteases can move through the intercellular spaces of Phaseolus cotyledons during germination to bring about hydrolysis of the storage proteins. They assume that later the products of hydrolysis - the amino acids and sugars - will themselves be transported through these intercellular channels to the developing seedling apex. It is of some importance to establish whether /Mectins occupy similar sites during germination, and work is in progress to determine the fate of lectins during cotyledon development in both jack bean and red kidney bean.

Role of lectins in plant cells The present results indicate that lectins with quite different substrate specificity, and presumably function, occupy different cellular sites in legume cotyledons. The biologically active lectins, Con A and PHA, apparently occur exclusively in cyto- plasmic sites. In contrast, the /?-Iectins, defined on their ability to precipitate the Yariv artificial /?-glycosyl antigens, are associated with the cell membrane and concentrated in the cell walls and intercellular spaces. We suggested earlier that some lectins may function as self-recognition factors in plant cells, determining the relationships between vegetative cells in tissues and organs. To function in this way, they pre- sumably need to be located in extracellular sites in the cell walls, or at the cell mem- brane. This has been found to be the case for the pollen-wall proteins which function in recognition reactions on the female stigma (see reviews by Knox, Heslop-Harrison & Helsop-Harrison, 1975; Heslop-Harrison, Knox, Heslop-Harrison & Mattsson, 1975). The /?-lectins certainly appear to be strategically sited to fulfil some role in cell communication in cotyledon cells. Jermyn & Yeow (1975) have demonstrated their widespread occurrence throughout the plant kingdom, and in various tissues and organs of individual plants. This suggests such a communication role need not be confined to cotyledons. Several other lectins are not exclusively located in seeds (see Lis & Sharon, 1973). Mialonier et al. (1973) record that an immunologically related form of PHA occurs in the young shoots of Phaseolus vulgaris. occurs in leaves, stems and roots of Phytolacca americana, sometimes in even higher concentrations than in the seed (Fames, Barker, Brownhill & Fanger, 1964). Hamblin & Kent (1973) have preliminary evidence for the presence of PHA on young root primordia acting as binding sites for symbiotic bacteria. SBA has recently been shown to have a similar Lectin localization in cotyledons 163 function in soy beans (Bohlool & Schmidt, 1974). Pseudomonas and other leaf-infecting micro-organisms may enter the host leaf via the intercellular spaces, and suscepti- bility or resistance - a recognition event - is determined from these sites (Nelson & Dickey, 1970). The/?-lectins so far examined comprise about 80% carbohydrate and 20% protein, so it is not inconceivable that they could carry determinants controlling both self-recognition and disease resistance. As Burnet (1971) pointed out in relation to the human immune system, it is likely that defence mechanisms have evolved as secondary elaborations to a basic system evolved for self-recognition. The present observations of the extracellular localization of certain lectins are consistent with such a role. This work is supported by a grant to R.B.K. from the Australian Research Grants Com- mittee, and was carried out during the tenure by A.E.C. of a University of Melbourne Research Fellowship. We thank Professors J. Heslop-Harrison and B. A. Stone for helpful comments on the manuscript, and Miss Eve Muchnicky, B.Sc, for her helpful assistance.

REFERENCES BERNHARD, W. & AVRAMEAS, S. (1971). Ultrastructural visualization of cellular carbohydrate components by means of concanavalin A. Expl Cell Res. 64, 232-236.' BOHLOOL, B. B. & SCHMIDT, E. L. (1974). Lectins: a possible basis for specificity in the Rhizobium-legume root nodule symbiosis. Science, N.Y. 185, 269—271. BURNET, F. M. (1971). 'Self-recognition' in colonial marine forms and flowering plants in relation to the evolution of immunity. Nature, Lond. 232, 230-235. FARNES, P., BARKER, B. E., BROWNHILL, L. E. & FANGER, H. (1964). Mitogenic activity in Phytolacca americana (pokeweed). Lancet ii, Nov. 21, 1100-1101. FEDER, N. & O'BRIEN, T. P. (1968). Plant microtechnique: some principles and new methods. Am. J. Bot. 55, 123-134. FLINT, F. O. & Moss, R. (1970). Selective staining of protein and starch in wheat flour and products. Stain Technol. 45, 75-79. GLIMELIUS, K., WALLIN, A. & ERIKSSON, T. (1974). Agglutinating effects of concanavalin A on isolated protoplasts of Daucus carota. Physiol. PL 31, 225-230. HAMBLIN, J. & KENT, S. P. (1973). Possible role of phytohaemagglutinin in Phaseolus vulgaris L. Nature, New Biol. 245, 28-30. HESLOP-HARRISON, J., KNOX, R. B., HESLOP-HARRISON, Y. & MATTSSON, O. (1975). Pollen- wall proteins: emission and role in incompatibility responses. In The Biology of the Male Gamete (ed. J. G. Duckett & P. A. Racey). Biol. J. Linn. Soc. 6, Suppl. 1 (in Press). JERMYN, M. A. (1974). A class of lectins widespread in the seeds of flowering plants. Proc. Aust. biochem. Soc. 7, 32. JERMYN, M. A. & YEOW, Y. M. (1975). A class of lectins present in the tissues of flowering plants. Aust. J. Plant Physiol. 2 (in Press). KNOX, R. B. (1975). Cell recognition and pattern formation in plants. In A Textbook of Develop- mental Biology (ed. P. F. Wareing & C. Grahame), ch. 10. Oxford: Blackwell (in Press). KNOX, R. B., HESLOP-HARRISON, J. & HESLOP-HARRISON, Y. (1975). Pollen-wall proteins: localization and characterization of gametophytic and sporophytic fractions. In The Biology of the Male Gamete (ed. J. G. Duckett & P. A. Racey). Biol. J. Linn. Soc. 6, Suppl. 1 (in Press). Lis, H. & SHARON, N. (1973). The biochemistry of plant lectins (phytohaemagglutinins). A. Rev. Biochem. 42, 541-574. MIALONIER, G., PRIVAT, J.-P., MONSIENY, M., KAHLEM, G. & DURAND, R. (1973)- Isolement, propri^tes physicochimique et localisation in vivo d'une phytoh&nagglutine (lectine) de Phaseolus vulgaris L. (var. rouge). Physiol. vig. 11, 519-537. NELSON, P. E. & DICKEY, R. S. (1970). Histopathology of plants infected with vascular bacterial pathogens. A. Rev. Phytopath. 8, 259-280. 164 A. E. Clarke, R. B. Knox and M. A. Jermyn OPIK, H. (1966). Changes in cell fine structure in the cotyledons of PhaseolusvulgarisL,. during germination. J. exp. Bot. 17, 427-439. So, L. L. & GOLDSTEIN, I. J. (1967). Protein and carbohydrate interaction. Application of the quantitative hapten inhibition test to polysaccharide-concanavalin A interaction. J. Ivimun. 99, 158-163. YARIV, J., RAPPORT, M. M. & GRAF, L. (1962). The interaction of glycosides and saccharides with antibody to the corresponding phenylazo glycosides. Biochem. J. 85, 383-388. YOMO, H. & TAYLOR, M. P. (1963). Histochemical studies on protease formation in the cotyledons of germinating bean seeds. Planta nz, 35-43.

{Received 20 January 1975)

Figs. 1, 2. Hand sections of 48-h-imbibed cotyledons of jack bean, Canavalia evsi- formis. Fig. 1. Localization of concanavalin A using FITC-labelled globulin, A, section through pair of cotyledons showing reaction of epidermal and parenchyma cells. Fluorescence (white in print) is intense in cytoplasmic sites, x 190. B, detail showing intense cytoplasmic fluorescence, especially around starch grains (s). x 300. c, control section treated with 10% a-methyl glucoside prior to staining with FITC-labelled globulin showing absence of cytoplasmic fluorescence, x 190. Fig. 2. Localization of /?-lectin in sections treated with /?-glucosyl Yariv antigen (1 mg/ml) in 10% DMSO. A, dense reaction product in intercellular spaces, and associated with plasma membrane on inner face of walls. Dark rings around starch grains due to refractivity. x 190. B, detail showing intercellular spaces filled with reaction product, x 480. c, intercellular space reaction product is associated with spherical vesicles, x 750. Lectin localization in cotyledons 165 166 A. E. Clarke, R. B. Knox and M. A. Jermyn

Figs. 3-6. Hand sections of 48-h-imbibed cotyledons of jack bean, Canavalia ensiformis. Fig. 3. Section treated with salicin (25 % in 0-15 M NaCl) for 15 min before staining with /?-glucosyl Yariv antigen (1 mg/ml) in 10% DMSO. Staining is absent in the intercellular spaces, except within cell wall sites (arrows), x 190. Fig. 4. Section treated with a-galactosyl Yariv antigen (1 mg/ml) in 10% DMSO. No reaction product is present, x 190. Fig. 5. Section treated with oc-mannosyl Yariv antigen (1 mg/ml) in 10% DMSO. Reaction product is present in cytoplasmic sites but is absent from the intercellular spaces, x 300. Fig. 6. Section stained with Ponceau 2R for localization of total protein, showing dense staining reaction in intercellular spaces (arrows), and cytoplasmic sites, x 190. Figs. 7-9. Hand sections of 48-h-imbibed cotyledons of other beans, treated with /?-glucosyl Yariv antigen (1 mg/ml) in 10% DMSO. Fig. 7. Section of red kidney bean, Phaseolus vtdgaris, showing reaction product in intercellular spaces, and associated with plasma membrane, x 190. Fig. 8. Section of lima bean, Phaseolus lunatus; localization as in Fig. 7. x 190. Fig. 9. Section of pigeon pea, Cajanus cajan, showing similar localization pattern. Note that dark rings around starch grains are due to refractivity. x 190. Lectin localization in cotyledons 167